Low molecular weight heparin assay, system and reagent therefor

ABSTRACT

A method, kit, system and reagent for measuring low molecular weight heparin in a whole blood sample is provided which involves the use of a Factor Xa activator, such as Russell&#39;s Viper Venom, as the coagulation assay initiator.

BACKGROUND OF THE INVENTION

[0001] 1. Field of Invention

[0002] The present invention relates to a dry chemistry format assay formeasuring the low molecular weight heparin content of a whole bloodsample, and a system and reagent for performing such an assay.

[0003] 2. Discussion of the Background

[0004] Low molecular weight heparins (LMWHs) are a heterogeneous groupof antithrombotic drugs produced from unfractionated heparin (UFH) usingdiverse chemical and enzymatic processes. LMWHs, like UFH, exhibit ananticoagulant effect by complexing with antithrombin (AT) to inactivateseveral of the coagulation enzymes preventing fibrin formation. Ofthese, Factor Xa and thrombin (IIa) are the most responsive toinhibition. LMWHs, introduced as antithrombotic drugs in the mid-1980s,are now established as the drug of choice for surgicalthromboprophylaxis and are increasingly replacing UFH in the acutetreatment of venous thromboembolic disorders. The low molecular weightheparin, enoxaparin, increasingly is used in patients with unstableangina (UA) and non-Q-wave myocardial infarction (NQMI) (J. Fareed etal, Past, present and future considerations on low molecular weightheparin differentiation: an epilogue. Semin Thromb Hemost, 25 Suppl3:145-7 (1999), and J. Hirsh et al, Heparin and low-molecular-weightheparin: mechanisms of action, pharmacokinetics, dosing, monitoring,efficacy, and safety. Chest, Jan;119(1 Suppl):64S-94S (2001)), whotransition to percutaneous coronary intervention (PCI) (Lovenox(enoxaparin sodium) injection package insert, ©1998, rev. 01/2001).Although the activated partial thromboplastin time (aPTT) and activatedclotting time (ACT) are the most common methods used to monitor UFH,they are relatively insensitive to LMWHs, such as enoxaparin. Whilechromogenic anti-Xa assays are commonly used to monitor theconcentration of LMWHs, such assays provide an indirect measure of drugconcentration and results are not routinely available in a cardiaccatheterization laboratory setting.

[0005] The LMWHs have mean molecular weights between 4000 to 6000daltons, and they have less ability to inactivate thrombin compared toUFH. Each LMWH is a specific mixture often demonstrating a uniqueanti-Xa/anti-IIa ratio and signature anticoagulant profile. The resultis an anti-Xa/anti-IIa ratio of approximately 3 to 14:1 (depending onthe brand of LMWH, dosage, and route of administration) compared to the1:1 ratio observed with UFH^((Lovenox P.I.)). The LMWH, enoxaparin, hasa mean molecular weight of approximately 4,500 daltons and, given at adose of 1.5 mg/kg subcutaneously (SC), is characterized by a higherratio of anti-Factor Xa to anti-Factor IIa activity (mean±SD, 14.0+3.1)(based on areas under anti-Factor activity versus time curves) comparedto the ratios observed for heparin (mean±SD,1.22±0.13)^((Lovenox P.I.)). This is an important distinction becausethe ability to prolong the APTT and ACT is proportional to anti-IIaactivity. Chromogenic anti-Xa assays provide estimates of enoxaparinconcentration only in dilute, supplemented plasma and are not suitablefor point-of-care (POC) testing.

[0006] More recently, clinical trials have confirmed the safety andefficacy of the LMWH, enoxaparin sodium (Lovenox®, Clexane®), in themanagement of acute coronary syndromes (ACS) (J. Fareed et al.,Thromosis and Hemostasis, Supplement 3, Vol. 25, 3-4 (1999)).

[0007] Blood clotting reactions, in general, are employed as clinicalassays to measure the time required for the formation of a fibrin clot.Blood clotting assays are principally used for screening, diagnosis, andfor monitoring patients receiving anticoagulant therapy. There are manytypes of coagulation assays. These include prothrombin time (PT),partial thromboplastin time (PTT), activated partial thromboplastin time(APTT), fibrinogen assay, thrombin clotting time (TCT), activatedclotting time (ACT), etc.

[0008] Before performing conventional clotting tests, a blood sample iscollected in a tube or syringe containing anticoagulant (citrate). Theblood sample is centrifuged, and the plasma separated (e.g., bydecantation) from the red blood cells. A measured quantity (usually 0.1ml) of plasma is pipetted into the reaction vessel or cuvette. Ameasured amount of reagent is then added manually via pipette orautomatically by means of other volumetric delivery systems capable ofmetering a known, preset quantity of reagent. Alternatively, the samplecan be added to the reagent directly.

[0009] Typically, 0.2 ml of reagent is employed. The addition of thereagent initiates the reaction. Many existing blood clotting assayssuffer from at least one of the following disadvantages: difficulty inperformance, requirement of highly trained personnel, inaccuracy inmeasurement, reagent instability, large consumption of reagent, etc.

[0010] One solution to this problem was addressed in Oberhardt, U.S.Pat. No. 5,110,727, in which a dry reagent based reaction slide isprovided for performing coagulation assays quickly, accurately andsimply. Such tests are marketed by Pharmanetics, Inc.

[0011] The capacity of blood to clot, as well as to not clot, isdependent on a large number of enzymatic factors and cofactors. Theability of central clinical laboratories to reliably and convenientlyassay for LMWH in whole blood or plasma samples can be critical inmonitoring individuals in LMWH therapy. The blood coagulation system isdominated by sequential proteolytic activation reactions of inactiveprecursors, called zymogens. Forward clotting reactions are controlledby simultaneous activation of anticoagulant zymogens that serve to limitthe extent of clot formation and initiate the fibrinolytic system toresolve the clot.

[0012] There is thus a strongly felt need for a simple, facile andaccurate method for the performance of blood clotting assays, e.g., inmedical applications. Such a method should be based on a minimum numberof manipulations of either a sample or reagent. Ideally such a methodshould be easily utilized by persons without extensive clinicallaboratory training and should require no sample or reagent-containingsolution preparation. It should not suffer the problems associated withreagent instability and be very accurate. It should permit effectivemixing of sample and reagent. It should require only a very small amountof sample. And it should be able to perform automatic treatments of thesample, e.g., it should not require centrifugation of the blood sampleor any other off line cell separation process. Available clottingparameter assays likewise suffer salient disadvantages.

[0013] Since the tests currently used for LMWHs are chromogenic assaysrequiring isolation of derived plasma from whole blood samples andsignificant processing time for performing the assay, an assay is neededthat can quantitatively measure LMWHs quickly and easily, using wholeblood and be performed at the bedside, in order to provide rapiddeterminations of LMWH therapeutic levels.

[0014] Additionally, conventional chromogenic assays measure actuallevels of LMWH in the plasma sample, but do not reflect the actualclotting dynamics of the patient's blood. Since the clotting dynamicscan depend or be confounded by a variety of factors, a test is neededthat will correlate the clotting time with the amount of LMWH in thesample, and will also detect other possible problems in the clottingdynamics that are independent of the LMWH.

[0015] Clotting parameter assays are referred to herein as function andstructure-based assays in the broad realm of coagulation diagnosticswhich do not utilize clot formation or clot lysis processes to generateend points. Most of these assays utilize chromogenic syntheticsubstrates to quantify molecular markers or specific factors orcomponents associated with coagulation. These are typically functionalreaction based assays as opposed to most immunoassays which could detectthe same molecules but utilize structure recognition and may thereforestill identify inhibited components or defective components, neither ofwhich may be functional.

SUMMARY OF THE INVENTION

[0016] Accordingly, one object of the present invention is to provide animproved coagulation assay for LMWHs, particularly enoxaparin.

[0017] A further object of the present invention is to provide such anassay that can be performed using whole blood.

[0018] A further object of the present invention is to provide such anassay that is based on a dry chemistry format.

[0019] A further object of the present invention is to provide reagentsfor coagulation assays for measurement of LMWHs.

[0020] A further object of the present invention is to provide acoagulation based assay in dry chemistry format that relates clottingtime to LMWH levels in a whole blood sample, while remaining sensitiveto factors that can affect clotting time of the sample.

[0021] These and other objects of the present invention have beensatisfied by the discovery of assays, reagents, methods and kits formeasuring low molecular weight heparin concentration in a whole bloodsample using a coagulation cascade reaction and monitoring coagulationtimes or kinetics, wherein the coagulation reagent is a dry formatreagent comprising magnetic particles and a Factor Xa activator, such asRussell's Viper Venom.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Many attendant features of this invention will become readilyappreciated as the same becomes better understood by reference to thefollowing detailed description when considered in connection with theaccompanying drawings:

[0023]FIG. 1 provides a graphical depiction of the relevant portion ofthe coagulation cascade in blood.

[0024]FIG. 2 provides a graphical representation of the in vitroclotting time response of citrated whole blood at different levels ofthe preferred LMWH enoxaparin.

[0025]FIG. 3 provides a graphical representation of in vitro clottingtime response of citrated whole blood at varying levels of four otherLMWHs.

[0026]FIG. 4 shows the response of the preferred ENOX test and thecorresponding derived plasma anti-Xa values as a function of time fromenoxaparin administration.

[0027]FIG. 5 shows the relationship between the ENOX test clot times forCWB and STACHROM® LMWH anti-Xa IU/ml values (from derived plasma) in asmall clinical trial.

[0028]FIG. 6 shows the results of a study on depletion of variousfactors in blood samples on the ENOX test clot times.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention relates to a method for measuring lowmolecular weight heparin concentration on a whole blood sample,comprising:

[0030] (i) combining a first, whole blood, component of the assay with asecond component of the assay, wherein the second component comprises adry coagulation assay reagent arranged in a substantially flattenedconfiguration and containing magnetic particles distributedsubstantially homogeneously therethrough and comprising a factor Xaactivator, and wherein the resulting mixture is subjected to (ia) anoscillating magnetic field or (ib) a moving permanent magnetic field or(ic) a combination of an oscillating magnetic field and a stationarypermanent magnetic field or (id) a rotating magnetic field, whereby thecombining of the first component with the second component substantiallysimultaneously initiates movement of the magnetic particles and acoagulation assay measurement; and

[0031] (ii) monitoring movement induced in the magnetic particles by(ia) or (ib) or (ic) or

[0032] (id) to obtain the coagulation assay measurement, wherein thecoagulation assay measurement correlates to a concentration of lowmolecular weight heparin in the whole blood sample.

[0033] In the method of the present invention, the method is carried outin an element for performing the method, wherein the element comprises achannel structure defining a sample well and a reaction volume in fluidcommunication with each other, the reaction volume preferably containingthe second component. The channel structure preferably has a geometrycausing the first, whole blood, component placed in the sample well tobe drawn into and fill the reaction volume via capillary action,wherein, after the reaction volume is filled, the first, whole blood,component (now combined with the second component and forming aresulting reaction mixture) remains stationary therein.

[0034] The element further preferably a means for channeling light froman outside source to the reaction volume, such as those described in thePharmanetics patents. The method of the present invention furthercomprises using a means for detecting light scattered or absorbed orreflected from the reaction volume to monitor the reaction. Suitablemeans for detecting light are described in the Pharmanetics patents.Preferably, the reaction element is disposed in sufficiently closeproximity to a permanent magnet and to an electromagnet such that thepermanent magnet and the electromagnet provide a combination of anoscillating magnetic field and a stationary permanent magnetic field.More preferably, the element is situated between the permanent magnetand the electromagnet.

[0035] The present invention further relates to a method for measuringlow molecular weight heparin concentration on a whole blood sample,comprising:

[0036] (i) adding a whole blood sample to a sample well of an elementcomprising:

[0037] a channel structure defining the sample well and a reactionvolume in fluid communication with each other, wherein the reactionvolume is defined by an upper surface having attached thereto areflectance layer, comprising a semipermeable matrix wherein thereaction volume contains a measured amount of at least one drycoagulation assay reagent arranged in a substantially flattenedconfiguration and containing magnetic particles distributedsubstantially homogeneously therethrough, wherein a specific volume ofthe sample is drawn into the reaction volume by capillary action andcontacts, together with the semipermeable layer, the reagent to therebysubstantially simultaneously initiate a coagulation assay measurement;and

[0038] (ii) performing the coagulation assay measurement by measurementthe reflectance of the semipermeable layer, wherein the dry coagulationassay reagent comprises a Factor Xa activator.

[0039] The present invention further relates to a kit for measuring lowmolecular weight heparin concentration on a whole blood sample,comprising, in one or more containers, a permanent magnet, a timingmeans, and an element containing at least one dry coagulation assayreagent arranged in a substantially flattened format and containingmagnetic particles distributed substantially homogeneously therethrough,wherein the at least one dry coagulation assay reagent comprises aFactor Xa activator. Additionally, the present invention relates to asystem for measuring low molecular weight heparin concentration on awhole blood sample, comprising:

[0040] (i) an instrument with a means for temperature control, a meansfor producing an oscillating magnetic field or for moving a permanentmagnetic field, an illuminating means, and a photometric monitoringmeans; and

[0041] (ii) an element for performing the measuring, the elementcomprising a channel structure defining a sample well and reactionvolume in fluid communication with each other, the channel structurehaving a geometry causing a liquid sample placed in the sample well tobe drawn into and filling the reaction volume via capillary action, thereaction volume comprising at least one dry coagulation assay reagentarranged in a substantially flattened configuration and containingmagnetic particles distributed substantially homogeneously therethrough,wherein the at least one dry coagulation assay reagent comprises aFactor Xa activator.

[0042] The system of the present invention can further comprise atransfer pipette, preferably made of an essentially nonthrombogenicmaterial, and comprising a vented end. The transfer pipette ispreferably capable of being filled with a liquid sample by capillaryaction, and is capable of expelling the liquid sample by means ofpressure after covering or sealing the vented end. The instrument of thesystem further can comprise a heating means comprising a resistiveheater strip and a thermistor situated in close proximity to theelement. In the system of the present invention, the element ispreferably suitable for performing a whole blood coagulation assay, withthe channel structure having a geometry causing a blood sample placed inthe sample well to be drawn into and filling the reaction volume viacapillary action, wherein after the reaction volume is filled, the bloodsample remains stationary therein, and wherein the element furthercomprises an optically or magnetically encodable information means, orboth, capable of providing at least one of calibration, quality control,test parameter and patient information. The illuminating means of thesystem preferably includes one or more light sources to illuminate theelement. The photometric monitoring means preferably comprises one ormore detectors for photometrically monitoring chromogenic orchromomodulating species present in the reaction volume. Suitable meansfor the physical devices of the system of the present invention aredescribed in the Pharmanetics patents.

[0043] The present invention further preferably relates to a system formeasuring low molecular weight heparin concentration in a whole bloodsample, comprising:

[0044] (i) a reaction element comprising (1) a sample well for receivinga liquid sample and (2) a reaction chamber containing a dry coagulationassay reagent arranged in a substantially flattened configuration and inwhich is embedded, substantially homogeneously therethrough, magneticparticles;

[0045] (ii) the sample well and reaction chamber being in fluidcommunication through a transport zone of geometry such that a volume ofliquid sample placed in the sample well and corresponding to the volumeof the reaction chamber is transported from the sample well to thereaction chamber simultaneously;

[0046] (iii) means for optically monitoring the reaction chamber;

[0047] (iv) means for subjecting the reaction chamber to an oscillatingmagnetic field;

[0048] (v) whereby, when the sample is introduced into the reactionchamber, the dry coagulation assay reagent is solubilized and themagnetic particles are thereby freed to move in an oscillating patterninduced by the oscillating magnetic field, thus providing a measurementof the kinetics of the coagulation assay corresponding to changes in thedegree of the magnetic particles movement relative to the oscillatingmagnetic field, wherein the dry coagulation assay reagent comprises aFactor Xa activator.

[0049] The system preferably further comprises a means for controllingthe moment transport of the liquid sample from the sample well to thereaction chamber is initiated. Suitable such means are described in thePharmanetics patents. The system of the present invention can furthercomprise a plurality of reaction chambers in fluid communication withthe sample well, and means for transporting a whole blood or plasmasample from one of the plurality of reaction chambers to another of theplurality of reaction chambers. In such an arrangement, it is possibleto have the patient sample be split among the plurality of reactionchambers, with each reaction chamber having different coagulationreagents present for monitoring different aspects of the patients blood.Suitable such plural reaction chamber elements are likewise described inthe Pharmanetics patents.

[0050] The present invention further relates to a method for measuringlow molecular weight heparin concentration in a whole blood sample,comprising:

[0051] (i) subjecting to an oscillating magnetic field a reactionelement bearing (1) a sample well for receiving a whole blood sample and(2) a reaction chamber containing a dry coagulation assay reagentarranged in a substantially flattened format and in which is embedded,substantially homogeneously therethrough, magnetic particles, the samplewell and reaction chamber being in fluid communication through atransport zone of geometry such that a volume of sample placed in thesample well and corresponding to the volume of the reaction chamber istransported from the sample well to the reaction chamber simultaneously;

[0052] (ii) adding the whole blood sample susceptible to coagulation tothe sample well whereby at least a part of the sample is introducedsimultaneously to the reaction chamber, the reagent is solubilized andthe particles are freed to move in an oscillating pattern induced by theoscillating magnetic field; and

[0053] (iii) optically monitoring the reaction chamber to measurekinetics for the coagulation assay corresponding to changes in thedegree of the particle movement relative to the magnetic field, whereinthe dry coagulation assay reagent comprises a Factor Xa activator.

[0054] The present invention relates to an assay for a LMWH, inparticular for enoxaparin (ENOX), in a whole blood sample using a drychemistry format reagent. Preferable assay elements (such as test cards)and their methods of preparation are described in U.S. Pat. Nos.4,849,340; 5,110,727; 5,350,676; 5,508,521; 5,601,991; 5,658,723;5,670,329; 5,677,133; 6,165,795; and 6,197,494, the entire contents ofwhich are hereby incorporated by reference (hereafter referred to as the“Pharmanetics patents”). However, the assay can be performed using assayelements of other types, as described below.

[0055] The present invention assay is designed to provide rapid resultsof the anticoagulant effect provided by LMWH, preferably enoxaparinsodium (Lovenox®, Clexane®). It is a one-step coagulation methodperformed on the Rapidpoint Coag analyzer, available from Bayer Corp. oron the TAS analyzer, available from Pharmanetics, Inc. The test utilizescitrated or non-citrated whole blood. Like other test devices describedin the Pharmanetics patents, the LMWH test is a dry chemistry test card.All of the components necessary to perform the test, with the exceptionof the patient sample, are included within the reaction chamber of thecard.

[0056] In the assay of the present invention, factor X is rapidlyconverted to factor Xa by the reagent containing a factor Xa activator,such as Russells' viper venom (RVV-X), initiating the clotting process,see FIG. 1. The assay element, preferably a test card as described inthe Pharmanetics patents, contains the Factor Xa activator, but allclotting factors necessary for test function (Factor X and V,prothrombin, fibrinogen, and antithrombin) are supplied by the patient'ssample. The assay measures the combined anti-Xa and anti-IIa activity ofthe low molecular weight heparin and is designed to measure clot timesin citrated whole blood (CWB) over a broad range of comparablespiked-CWB derived plasma activities. Conventional tests for LMWH areonly applicable up to about a range of anti-Xa activities from 0.0 to1.0 anti-Xa IU/ml. However, the present assay provides the combinedanti-Xa and anti-IIa activity of the LMWH and measures clot times in CWBcomparable to a spiked-CWB derived plasma with an anti-Xa range of 0.0to 3.0 anti-Xa IU/ml LMWH. The results generated by the present assayare indicative of the overall anticoagulant effect produced by the LMWHin whole blood.

[0057] The reagent comprises magnetic particles and a factor Xaactivator. Suitable factor Xa activators include various Xa activatingenzymes derived from snake venoms, including but not limited toRussell's Viper Venom (RVV-X), Vipera aspis aspis, Bothrops atrox,Saw-scaled viper Echis carinatus venom, and Cerastes cerastes venom.

[0058] The LMWH assay reagent of the present invention may furthercomprise one or more members selected from the group consisting ofbuffers, lyophilization aids, non-ionic detergents and proteins.Suitable buffers include, but are not limited to, HEPES, TRIS, and PIPESin the pH range of from 6.0 to 8.0, more preferably from pH 6.0-7.0,most preferably from pH 6.3-6.8. Preferred lyophilization aids include,but are not limited to, sucrose, lactose, mannitol and trehalose, withtrehalose being most preferred. Non-ionic detergents are preferably oneor more polysiloxanes combined with one or more detergents selected fromPluronic® surfactants, and PEO/PPO block copolymers. These componentsare preferably used in a combination of 1-10wt % polysiloxane and 90-99wt % of the Pluronic® or PEO/PPO block copolymer, most preferably in a1:99 ratio of polysiloxane: Pluronic® or PEO/PPO block copolymer. Thepolysiloxanes used therein can be any polysiloxane type detergent,preferably polydimethylsiloxane PDMS detergents. The proteins useful inthe present reagent preferably include, but are not limited to bovineserum albumin (BSA) and ovalbumin, with BSA being most preferred.

[0059] In the present assay, factor X is rapidly converted to factor Xaby the factor Xa activator, initiating the clotting process. The LMWH,preferably enoxaparin, from the patient's blood, complexes withantithrombin, to inhibit factor Xa and lengthen the clotting time in adose-dependent manner. The reported clotting time increases in a dosedependent manner to the LMWH concentration present in the sample. Theresults generated by the test are indicative of the anticoagulant effectproduced by the LMWH in whole blood.

[0060] Additionally, the results of the present assay can be used todetermine if a patient is responding normally to LMWH therapy. Forexample, in certain patients, the blood is lacking in antithrombin, anessential factor needed for the anticoagulant effect of UFH or LMWH towork. When such a patient is monitored using conventional chromogenicLMWH assays, the assay returns only the level of LMWH, withoutreflecting the clotting dynamics of the patient's coagulation system.However, with the test of the present invention, one obtains a clottingtime measurement that will readily show the physician that the level ofLMWH is having little or no effect on the patient, since their clottingtime will be relatively unchanged upon addition of increasing levels ofLMWH. This will quickly tell the physician that there is another issueat play in the patient's anticoagulation status, thereby saving precioustime in modifying the therapy. Using the conventional chromogenic tests,such results would require two or more tests to obtain and could easilybe missed.

[0061] The test card of the present invention can be used to monitor theeffects of the low molecular weight heparin (LMWH), preferably ofLovenox®/Clexane® (enoxaparin, sodium), in citrated or non-citratedwhole blood.

[0062] The test provides information on patient's whole blood responseto LMWH, such as enoxaparin by measurement of the clotting time using afactor Xa activated clotting method. To the present inventors'knowledge, the present assay provides the first assay for LMWH that canbe accurately performed using whole blood.

[0063] In the present invention, the magnetic particles are induced tomove by being subjected to either (I) an oscillating magnetic field or(2) a moving permanent magnetic field or (3) a combination of anoscillating magnetic field and a stationary permanent magnetic field, or(4) a rotating magnetic field. The movement of the magnetic particles isthen monitored in the performance of the assay. The magnetic field ofthe present invention can be generated using any of the magnetic fieldgenerating means described in the Pharmanetics patents. The movement ofthe magnetic particles is preferably detected and analyzed also asdescribed in the Pharmanetics patents.

[0064] The clotting assays of this invention are performed on a reactionelement. This reaction element can be any element which will support thereagents used in the assay and permit monitoring movement of themagnetic particles. Such reaction elements include microtiter plates,their equivalents, substantially flat surfaces or the reaction slideprovided by the Pharmanetics patents.

[0065] The magnetic particles of the present invention assays arepresent in an amount of 0.5, or lower, to 50 milligrams of magneticparticles, preferably 1 to 10 milligrams, per milliliter of dry reagent.

[0066] Examples of this invention are set forth below. However, it is tobe understood that these examples are given by way of illustration onlyand are not to be construed as limiting the invention either in spiritor in scope, as many modifications both in doses and methods could bepossible to those skilled in the art.

EXAMPLES

[0067] Dose Response for Enoxaaparin embodiment of present assay An invitro dose-response relationship between mean ENOX CWB clotting timesfor 10 individuals and the derived plasma enoxaparin concentrations isshown in FIG. 2 and TABLE 1. TABLE 1 Combined In Vitro Response ofRapidpoint ® Coag ENOX Test to Enoxaparin in CWB from 10 IndividualDonors Mean Anti-Xa* 0.0 0.38 0.82 1.92 3.0 Mean C.T. 122.8 203.1 256.8320.7 380.7 SD 21.1 24.9 27.3 34.7 40.2

[0068] Whole blood from a normal adult was drawn into a Vacutainer brandsample collection tube containing 0.105M (3.2%) sodium citrate at theratio of 9 parts blood to 1 part citrate. After mixing the tube bygentle inversion, the citrated whole blood was aliquoted into plastictubes and supplemented with the low molecular weight heparin (LMWH),Lovenox® (enoxaparin sodium) to yield five whole blood enoxaparinsolutions with nominal concentrations ranging from about 0.0 to about3.0 IU/ml enoxaparin. Each sample was tested once on a dry-chemistryLMWH test. The whole blood solutions were centrifuged within 5 minutesto obtain platelet poor plasma. The platelet poor plasma was then testedfor enoxaparin concentration using the chromogenic STACHROM® LMWH assay(catalog #00906; Parsippany, N.J.), performed on an Electra 900Canalyzer (Medical Laboratory Automation, Inc., Pleasantville, N.Y.). Theresults were calibrated to a preparation of enoxaparin (Aventis UNWSD3075). This procedure was repeated for a total of ten normal adults.Individual results and the average for all 10 individuals is shown inTable 2 and FIG. 2. TABLE 2 In Vitro Response of the Low MolecularWeight Heparin (LMWH) Test to enoxaparin in Whole Blood R (correlation)Enoxaparin concentration* 0.00 0.32 0.76 2.29 2.98 Donor 1 LMWH Test142.7 222.1 253.0 312.3 444.8 0.951 Clotting Time (seconds) Enoxaparinconcentration* 0.00 0.37 0.78 1.94 2.75 Donor 2 LMWH Test 127.6 193.8259.6 346.8 405.2 0.981 Clotting Time (seconds) Enoxaparinconcentration* 0.00 0.33 0.75 1.67 3.29 Donor 3 LMWH Test 147.7 259.1314.8 396.0 420.1 0.872 Clotting Time (seconds) Enoxaparinconcentration* 0.00 0.35 0.75 1.67 2.32 Donor 4 LMWH Test 104.8 196.8260.5 309.6 351.0 0.944 Clotting Time (seconds) Enoxaparinconcentration* 0.00 0.41 0.81 1.64 2.70 Donor 5 LMWH Test 139.0 214.8270.1 316.6 422.8 0.983 Clotting Time (seconds) Enoxaparinconcentration* 0.00 0.37 0.80 1.82 3.16 Donor 6 LMWH Test 152.0 206.8272.0 350.8 381.1 0.942 Clotting Time (seconds) Enoxaparinconcentration* 0.00 0.46 0.88 2.18 3.30 Donor 7 LMWH Test 101.2 192.0238.2 290.8 337.2 0.938 Clotting Time (seconds) Enoxaparinconcentration* 0.00 0.44 0.93 2.23 3.32 Donor 8 LMWH Test 111.1 196.6252.6 307.6 365.5 0.95 Clotting Time (seconds) Enoxaparin concentration*0.00 0.37 0.87 1.82 2.96 Donor 9 LMWH Test 101.2 177.8 236.1 297.8 348.90.958 Clotting Time (seconds) Enoxaparin concentration* 0.00 0.41 0.821.97 3.04 Donor 10 rep1 101.0 171.3 210.8 278.2 330.5 0.972 AverageEnoxaparin 0.00 0.38 0.82 1.92 2.98 Concentration Average LMWH Test122.8 203.1 256.8 320.7 380.7 0.963 Clotting Time (seconds) STDEV 21.124.9 27.3 34.7 40.2

[0069] These results show an increasing clotting time to increasingconcentrations of enoxaparin and good correlation to the laboratoryreference test.

[0070] The mean correlation (r) of the 10 individual dose-responserelationship was 0.963.

[0071]5

[0072] Preferred Enoxaparin embodiment A small clinical trial wasconducted at four centers with consenting patients primarily receivingenoxaparin for treatment of acute coronary syndromes and/or percutaneouscoronary intervention (PCI) (n=35) and secondarily those receivingenoxaparin for prevention of deep venous thrombosis (DVT) during totalknee/hip replacement (TKHR)(n=8). Two to four samples were collectedfrom TKHR patients receiving subcutaneous (30 mg-BID SC) dosing withenoxaparin. Samples were collected to reflect peak anti-Xa activity (3-5hours after dose) and at the nadir (just prior to dosing) to obtainsamples that span the entire range of anti-Xa activity. At least 3samples were collected in patients undergoing PCI. PCI patients receiveddrug via intravenous (IV) bolus (0.75 mg/kg) at the start of theprocedure (D. J. Kereiakes et al., Combination enoxaparin and abciximabtherapy during percutaneous coronary intervention: “NICE guys finishfirst”. J Invasive Cardiol Feb;12 Suppl A:IA-5A, (2000)). Samples werecollected pre-drug administration (baseline), peak (5-15 minutes afterbolus), therapeutic range (45-60 minutes after bolus), and then justprior to pulling the sheath (approx. 8-10 hours after bolus). Patientsreceiving UFH, direct thrombin inhibitors, and vitamin K antagonistswere not eligible for this study. Batch anti-Xa determinations wereperformed using the STACHROM® LMWH assay (Diagnostica Stago, Parsippany,N.J.) on an Electra 900C analyzer (Medical Laboratory Automation, Inc.,Pleasantville, N.Y.) which measures anti-Xa activity using theamidolytic method. The results were calibrated to a standard preparationof enoxaparin (L/N WSD3075 Aventis Pharmaceuticals, Inc., Strasbourg67917, France). Samples were collected at four sites from 31 male and 12female patients. Demographics, date of birth, sex, age, height, weight,and total body surface area (TBSA) of each patient are found in TABLE 3.The primary indication for hospitalization at Site 3 was for knee andhip replacement surgery while at the other sites the indication was forPCI. Treatment information at the four contributing sites is summarizedin Table 4. The inclusion of IV administered enoxaparin achieved anti-Xalevels from 1-2 anti-Xa IU/ml in derived plasma. A total of 116 samplesfrom 43 patients were included in this study. TABLE 3 Demographics ofthe Enoxaparin Test Clinical Trial Gender Site Female Male Total Age(Yrs) Height (cm) Weight (kg) TBSA (m2) 1 4 16 20 Mean 67.4 173.2 83.42.00 Max 83 188.0 111.2 2.41 Min 48 150.0 53.0 1.49 2 3 4 7 Mean 65166.1 88.5 2.02 Max 80 185.0 102.1 2.19 Min 54 129.5 77.7 1.69 3 2 6 8Mean 60.5 164.1 70.2 1.78 Max 69 190.5 96.0 2.25 Min 37 152.4 50.0 1.484 3 5 8 Mean 63.3 169.3 73.8 1.85 Max 78 185.0 93.0 2.16 Min 51 157.042.2 1.36 All 12 31 43 Mean 64.9 169.6 80.0 1.93 Sites Max 83 190.5111.2 2.41 Min 37 129.5 42.2 1.36

[0073] TABLE 4 Dosing Regimes from Sites and Primary Indication SitesTreatment Primary Indication 1 0.75 mg/kg IV PCI/PTCA 2 0.75 mg/kg IVPCI 3 30 mg qd SC Orthopedic Surgery 4 0.73 to 1.07 mg/kg IV PCI

[0074] Clotting times (CT) from citrated whole blood CWB samples rangedfrom 80 to 470 seconds as measured by the Rapidpoint® ENOX test.Fifty-one samples (44%) had CT of 199 seconds or less, 36 samples (31%)had a CT of 200 to 300 seconds, and 29 samples (25%) had a CT of greaterthan 300 seconds. The population coefficient of variation (CV) forduplicate clot time measurements was 8.8% in whole blood. Thesecoefficients of variation are acceptable. Corresponding derived-plasmaenoxaparin concentrations ranged from 0.0 to 1.8 anti-Xa IU/ml.Distribution of enoxaparin concentrations observed in the plasma derivedfrom these clinical samples (n=116) was 41 samples (36%)<0.1 IU/ml, 49samples (43%) 0.1 tc 1.0 IU/ml, and 26 samples (22%) 1.1 to 1.8 IU/ml asmeasured by Stago Stachrom anti-Xa assay. The population coefficient ofvariation for the STACHROM® (LMWH method using duplicate anti-Xa IU/mlmeasurements was 9.4%. This relatively high CV may be due to theadditional error introduced by dilution of samples that containenoxaparin concentrations beyond the range of the STACHROM® LMWH test(This range is 0.01 ˜1.0 anti-Xa IU/ml).

[0075] The relationship between the ENOX test clot times for CWB andSTACHROM® LMWH anti-Xa IU/ml values (from derived plasma) are shown inFIG. 5. Correlation between the ENOX test card clot times and theSTACHROM® LMWH results is shown with linear regression and has acorrelation coefficient (r) of 0.805. A similar relationship between theENOX test and the STACHROM® LMWH was observed if the results ofindividual clot time measurements were compared to the single STACHROM®LMWH anti-Xa IU/ml measurement (r=0.777). Overall, the trial provided anappropriate concentration range of enoxaparin containing samples forevaluation of the ENOX test card, especially for PCI patients in therange of 0.6 to 1.8 anti-Xa IU/ml of enoxaparin (J. P. Collet et al,Percutaneous coronary intervention after subcutaneous enoxaparinpretreatment in patients with unstable angina pectoris. Circulation Feb6;103(5):658-63 (2001)).

[0076]FIG. 4 shows the response of the ENOX test and the correspondingderived plasma anti-Xa values as a function of time from enoxaparinadministration. The two sets of data mirror each other with the peak invivo enoxaparin concentration occurring within 10 minutes of injectionand ENOX test follows the pharmacokinetics of enoxaparin.

[0077] INTERFERENCE STUDIES

[0078] A. Deficient plasma studies In vitro studies were performed withnormal pooled and affinity-depleted plasmas to determine the factorsensitivities of the test. At the plasma equivalent of 1.0 anti-Xa IU/mlenoxaparin clotting time increased 20% or more when levels offibrinogen, prothrombin, and Factor X decreased to levels of <10%, <10%,and 60% of normal, respectively. A representative summary of the studiesfor Factor X is shown in FIG. 6. A decrease in antithrombin of 90% ofnormal led to a decrease in clotting time of 25%. The ENOX test isdependent upon these critical coagulation factors in the common cascade.B. Drug Effects and Common Interferences In vitro experiments usingcitrated whole blood indicates the ENOX test is insensitive to lipids(to 20 mg/ml), antiplatelet agents (abciximab, eptifibatide, tirofiban,aspirin) and fibrinolytic agents (alteplase, tenecteplase), andhematocrit. Hemodilution with IsoLyte or saline to 15% did not affecttest results, Table 5. TABLE 5 Summary of Interference Testing ForRapidpoint ® ENOX Test Sample Interferent Tested EFFECT Type Lipid Noeffect 0-20 mg/ml CWB Hematocrit No effect 20-50% HCT CWB HemodilutionNo effect at 15% CWB Tirofiban No effect at 0-5600 ng/ml CWB Aspirin Noeffect at 0-300 ug/ml CWB Abciximab No effect at 0-3600 ng/ml CWBEptifibatide No effect at 0-2600 ng/ml CWB Ketorolac tromethamine Noeffect at 0-12 ng/ml CWB Alteplase No effect at 1000-3200 ng/ml CWBTenecteplase No effect at 0-10,000 ng/ml CWB Nitroglycerin No effect at0-1000 ng/ml CWB

[0079] Repeatability in preparation of assay and performance of assayThe within run CV values for 0.0 and 1.0 Iu/ml enoxaparin samples were6.3 and 5.5% and the lot-to-lot CV values for 0.0 and 1.0 IU/mlenoxaparin samples were 5.2 and 5.5%, respectively. Total imprecision at1.0 IU/ml enoxaparin was <7.0%.

[0080] It will be apparent from the above detailed description thatthere are many variations in the present invention and the same aredeemed to be subject to this invention as set forth in the appendedclaims.

1. A method for measuring low molecular weight heparin concentration ona whole blood sample, comprising: (i) combining a first, whole blood,component of the assay with a second component of the assay to form aresulting mixture, wherein said second component comprises a drycoagulation assay reagent arranged in a substantially flattenedconfiguration and containing magnetic particles distributedsubstantially homogeneously therethrough and comprising a factor Xaactivator, and wherein said resulting mixture is subjected to (ia) anoscillating magnetic field or (ib) a moving permanent magnetic field or(ic) a combination of an oscillating magnetic field and a stationarypermanent magnetic field or (id) a rotating magnetic field, whereby saidcombining of said first component with said second componentsubstantially simultaneously initiates movement of said magneticparticles and a coagulation assay measurement; and (ii) monitoringmovement induced in said magnetic particles by (ia) or (ib) or (ic) or(id) to obtain said coagulation assay measurement, wherein saidcoagulation assay measurement correlates to a concentration of lowmolecular weight heparin in the whole blood sample.
 2. The method ofclaim 1, wherein said whole blood sample is a citrated whole bloodsample.
 3. The method of claim 1, wherein said magnetic particles areinduced to move by applying an oscillating magnetic field thereto. 4.The method of claim 1, wherein said magnetic particles are induced tomove by applying a moving permanent magnetic field thereto.
 5. Themethod of claim 1, wherein said method is carried out in an element forperforming said method, said method comprising adding said first, wholeblood, component to said element, wherein said element comprises achannel structure defining a sample well and a reaction volume in fluidcommunication with each other, said reaction volume containing saidsecond component, said channel structure having a geometry causing saidfirst, whole blood, component placed in said sample well to be drawninto and filling said reaction volume via capillary action, wherein,after said reaction volume is filled, said first, whole blood, componentremains stationary therein.
 6. The method of claim 5, wherein saidelement further comprises a means for channeling light from an outsidesource to said reaction volume.
 7. The method of claim 6, furthercomprising using a means for detecting light scattered or absorbed orreflected from said reaction volume.
 8. The method of claim 7, whereinsaid element is disposed in sufficiently close proximity to a permanentmagnet and to an electromagnet such that said permanent magnet and saidelectromagnet provide said combination of an oscillating magnetic fieldand a stationary permanent magnetic field.
 9. The method of claim 8,wherein said element is situated between said permanent magnet and saidelectromagnet.
 10. The method of claim 1, wherein said magneticparticles are induced to move by application of a rotating magneticfield.
 11. The method of claim 1, wherein said Factor Xa activator isRussell's Viper Venom.
 12. The method of claim 1, wherein said lowmolecular weight heparin is enoxaparin.
 13. A method for measuring lowmolecular weight heparin concentration on a whole blood sample,comprising: (i) adding a whole blood sample to a sample well of anelement comprising: a channel structure defining the sample well and areaction volume in fluid communication with each other, wherein saidreaction volume is defined by an upper surface having attached thereto areflectance layer, comprising a semipermeable matrix wherein saidreaction volume contains a measured amount of at least one drycoagulation assay reagent arranged in a substantially flattenedconfiguration and containing magnetic particles distributedsubstantially homogeneously therethrough, wherein a specific volume ofsaid sample is drawn into said reaction volume by capillary action andcontacts, together with said semipermeable layer, said reagent tothereby substantially simultaneously initiate a coagulation assaymeasurement; and (ii) performing said coagulation assay measurement bymeasurement the reflectance of said semipermeable layer, wherein saiddry coagulation assay reagent comprises a Factor Xa activator.
 14. A kitfor measuring low molecular weight heparin concentration on a wholeblood sample, comprising, in one or more containers, a permanent magnet,a timing means, and an element containing at least one dry coagulationassay reagent arranged in a substantially flattened format andcontaining magnetic particles distributed substantially homogeneouslytherethrough, wherein said at least one dry coagulation assay reagentcomprises a Factor Xa activator.
 15. The kit of claim 14, furthercomprising a transfer pipette.
 16. The kit of claim 15, wherein saidtransfer pipette is made of an essentially nonthrombogenic material,comprises a vented end, is capable of being filled with a liquid sampleby capillary action, and is capable of expelling said liquid sample bymeans of pressure after covering or sealing said vented end.
 17. Asystem for measuring low molecular weight heparin concentration on awhole blood sample, comprising: (i) an instrument with a means fortemperature control, a means for producing an oscillating magnetic fieldor for moving a permanent magnetic field, an illuminating means, and aphotometric monitoring means; and (ii) an element for performing saidmeasuring, said element comprising a channel structure defining a samplewell and reaction volume in fluid communication with each other, saidchannel structure having a geometry causing a liquid sample placed insaid sample well to be drawn into and filling said reaction volume viacapillary action, said reaction volume comprising at least one drycoagulation assay reagent arranged in a substantially flattenedconfiguration and containing magnetic particles distributedsubstantially homogeneously therethrough, wherein said at least one drycoagulation assay reagent comprises a Factor Xa activator.
 18. Thesystem of claim 17, further comprising a transfer pipette.
 19. Thesystem of claim 18, wherein said transfer pipette is made of anessentially nonthrombogenic material, comprises a vented end, is capableof being filled with a liquid sample by capillary action, and is capableof expelling said liquid sample by means of pressure after covering orsealing said vented end.
 20. The system of claim 17, wherein saidinstrument further comprises a heating means comprising a resistiveheater strip and a thermistor situated in close proximity to saidelement.
 21. The system of claim 17, wherein said element is suitablefor performing a whole blood coagulation assay, said channel structurehaving a geometry causing a blood sample placed in said sample well tobe drawn into and filling said reaction volume via capillary action,wherein after said reaction volume is filled, said blood sample remainsstationary therein, and wherein said element further comprises anoptically or magnetically encodable information means, or both, capableof providing at least one of calibration, quality control, testparameter and patient information.
 22. The system of claim 17, whereinsaid illuminating means includes one or more light sources to illuminatesaid element and wherein said photometric monitoring means comprises oneor more detectors for photometrically monitoring chromogenic orchromomodulating species present in said reaction volume.
 23. A systemfor measuring low molecular weight heparin concentration in a wholeblood sample, comprising: (i) a reaction element comprising (1) a samplewell for receiving a liquid sample and (2) a reaction chamber containinga dry coagulation assay reagent arranged in a substantially flattenedconfiguration and in which is embedded, substantially homogeneouslytherethrough, magnetic particles; (ii) said sample well and saidreaction chamber being in fluid communication through a transport zoneof geometry such that a volume of liquid sample placed in said samplewell and corresponding to the volume of said reaction chamber istransported from said sample well to said reaction chambersimultaneously; (iii) means for optically monitoring said reactionchamber; (iv) means for subjecting said reaction chamber to anoscillating magnetic field; (v) whereby, when said sample is introducedinto said reaction chamber, said dry coagulation assay reagent issolubilized and said magnetic particles are thereby freed to move in anoscillating pattern induced by said oscillating magnetic field, thusproviding a measurement of the kinetics of said coagulation assaycorresponding to changes in the degree of said magnetic particlesmovement relative to said oscillating magnetic field, wherein said drycoagulation assay reagent comprises a Factor Xa activator.
 24. Thesystem of claim 23, further comprising a means for controlling themoment transport of said liquid sample from said sample well to saidreaction chamber is initiated.
 25. The system of claim 23, furthercomprising a plurality of reaction chambers in fluid communication withsaid sample well, and means for transporting a whole blood or plasmasample from one of said plurality of reaction chambers to another ofsaid plurality of reaction chambers.
 26. A method for measuring lowmolecular weight heparin concentration in a whole blood sample,comprising: (i) subjecting to an oscillating magnetic field a reactionelement bearing (1) a sample well for receiving a whole blood sample and(2) a reaction chamber containing a dry coagulation assay reagentarranged in a substantially flattened format and in which is embedded,substantially homogeneously therethrough, magnetic particles, saidsample well and reaction chamber being in fluid communication through atransport zone of geometry such that a volume of sample placed in saidsample well and corresponding to the volume of said reaction chamber istransported from said sample well to said reaction chambersimultaneously; (ii) adding the whole blood sample susceptible tocoagulation to said sample well whereby at least a part of said sampleis introduced simultaneously to said reaction chamber, said reagent issolubilized and said particles are freed to move in an oscillatingpattern induced by said oscillating magnetic field; and (iii) opticallymonitoring said reaction chamber to measure kinetics for the coagulationassay corresponding to changes in the degree of said particle movementrelative to said magnetic field, wherein said dry coagulation assayreagent comprises a Factor Xa activator.